GBS bacteria are a recognized etiological agent for bacteremia and/or meningitis in infants, and for infections in adults. Baker, "Group B Streptococcal Infections" in Advances in Internal Medicine, 25:475-500 (1980). Accordingly, it is important to develop rapid and definitive assays for diagnosis of GBS infection, and methods of generating protection against GBS, particularly in infants and compromised individuals.
The capsular polysaccharides from GBS bacteria are known to be important to GBS virulence and the development of protective immunity. See Kasper et al. U.S. Pat. No. 5,302,386. Moreover, the CP of recognized GBS types (I-V) are chemically related but antigenically distinct having repeating structures composed of galactose, glucose, N-acetyl glucosamine, and N-acetyl-neuraminic (sialic) acid.
Infants and young children have poor immunogenic response to polysaccharide antigens. These responses are characterized as being T cell independent and therefore are not associated with important attributes such as memory, isotype switching, or affinity maturation, which are necessary for conferring long term immunologic protection against subsequent infection. To circumvent this lack of an effective immunogenic response in infants and young children to polysaccharides, the art has developed means of converting the T cell independent response to T cell dependent response by covalently coupling polysaccharide bacterial antigens to a carrier protein to form a conjugate molecule. See, Jennings et al. U.S. Pat. No. 4,356,170, which is incorporated herein by reference.
Various procedures have been described in the art for conjugating capsular polysaccharides to proteins. For review, see Contributions to Microbiology and Immunology, vol 10, Conlugate Vaccines, volume editions J. M. Cruse and R. E. Lewis, Jr., 1989, which is incorporated herein by reference. In one method, polysaccharide is subjected to mild acid hydrolysis to produce reducing end groups capable of reacting with protein to form a covalent bond. Anderson, P. A., Infect. Immun., 39:233-238 (1983). However, the terminal sugar groups which participate in conjugating to protein exist in equilibrium between a hemiacetal and aldehyde and therefore couple to protein with poor efficiency. To overcome the poor reactivity of the terminal reducing sugar, the art turned to mild oxidation to introduce stable aldehyde groups at terminal positions of polysaccharides used to conjugate to protein. Jennings et al. U.S. Pat. No. 4,356,170, supra.
Kasper et al. U.S. Pat. No. 5,302,386 and International application WO 94/06467, respectively relate to GBS type III and II conjugate vaccines, both of which are incorporated herein by reference. According to the U.S. Pat. No. 5,302,386, endo-.beta.-galactosidase is used to cleave the polysaccharide backbone to produce products suitable for conjugating to protein. Oxidation of at least two terminal sialic acid groups to produce cross-linked conjugates is described in the WO 94/06467 application.
Type III GBS capsular polysaccharides are composed of a backbone of repeating branched pentasaccharide units. Jennings et al., Canadian J. Biochem., 58:112-120 (1980). One study of type III GBS polysaccharides reports that the natural immunodeterminant site is located at the side chain-backbone junction. Jennings et al., Biochemistry, 20:4511-4518 (1980). The presence of the side chain terminal N-acetyl-neuraminic acid residues reportedly was critical for immunodeterminant expression.
Prior methods of depolymerizing GBS II or III polysaccharides rely on either costly enzymatic methods or on acid hydrolysis which may alter the antigenicity of the CP due to removal of the labile terminal sialic acid groups. Accordingly, there is a need for relatively inexpensive and mild chemical procedures which are effective for depolymerizing GBS type II and III CP in a manner which results in fragments which are useful for producing CP-protein conjugate vaccines.